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1.
Nat Commun ; 12(1): 4884, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385460

RESUMO

Pathology is practiced by visual inspection of histochemically stained tissue slides. While the hematoxylin and eosin (H&E) stain is most commonly used, special stains can provide additional contrast to different tissue components. Here, we demonstrate the utility of supervised learning-based computational stain transformation from H&E to special stains (Masson's Trichrome, periodic acid-Schiff and Jones silver stain) using kidney needle core biopsy tissue sections. Based on the evaluation by three renal pathologists, followed by adjudication by a fourth pathologist, we show that the generation of virtual special stains from existing H&E images improves the diagnosis of several non-neoplastic kidney diseases, sampled from 58 unique subjects (P = 0.0095). A second study found that the quality of the computationally generated special stains was statistically equivalent to those which were histochemically stained. This stain-to-stain transformation framework can improve preliminary diagnoses when additional special stains are needed, also providing significant savings in time and cost.


Assuntos
Biópsia com Agulha de Grande Calibre/métodos , Aprendizado Profundo , Diagnóstico por Computador/métodos , Nefropatias/patologia , Rim/patologia , Coloração e Rotulagem/métodos , Algoritmos , Corantes/química , Corantes/classificação , Corantes/normas , Diagnóstico Diferencial , Humanos , Nefropatias/diagnóstico , Patologia Clínica/métodos , Patologia Clínica/normas , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/normas
2.
ACS Appl Mater Interfaces ; 13(33): 40070-40078, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34387999

RESUMO

Aminothiols are closely related to chronic kidney disease, but little is known regarding levels of related aminothiols in the urine of immunoglobulin A vasculitis with nephritis (IgAVN) patients. Herein, a well-defined core-shell Zr-based metal-organic framework (Zr-MOF) composite SiO2@50Benz-Cys was constructed as a mercury ion affinity material via a solvent-assisted ligand exchange strategy for the selective extraction and enrichment of low-concentration aminothiols in IgAVN patient urine. SiO2@50Benz-Cys was competent to enrich the total glutathione (GSH) and total homocysteine (Hcy) in virtue of the excellent affinity after chelation with mercury ions. The extraction efficiencies were closely related to the pH, dithiothreitol amount, and the dose of functional Zr-MOF. Coupled with HPLC-MS/MS in optimized conditions, GSH and Hcy were determined with low detection limits of 0.5 and 1 nmol L-1, respectively. The recoveries of GSH and Hcy for the urine sample at three spiked levels were in the range of 85.3-105% and 79.5-103%, which showed good precision and accuracy. Benefiting from the matrix interference elimination in the process of extraction, the simultaneous detection of aminothiols in the urine of the healthy group and immunoglobulin A vasculitis (IgAV) and IgAVN patients was successfully carried out, suggesting that the Zr-MOF and the robust method together provided a potential application in the analysis of urinary biomolecules. The analysis of variance (ANOVA) showed that the levels of GSH and Hcy had significant differences between the patients and the control. This work is very valuable as it provides a better understanding of concentration alterations of GSH and Hcy in urine involved with IgAVN for clinical research.


Assuntos
Glutationa/urina , Homocisteína/urina , Estruturas Metalorgânicas/química , Nefrite/diagnóstico , Zircônio/química , Técnicas Biossensoriais , Cromatografia Líquida de Alta Pressão , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dióxido de Silício , Coloração e Rotulagem/métodos , Compostos de Sulfidrila/química , Espectrometria de Massas em Tandem
3.
Methods Mol Biol ; 2350: 253-265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331290

RESUMO

Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.


Assuntos
Corantes Fluorescentes , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Microscopia de Fluorescência/métodos , Estrutura Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Peixe-Zebra
4.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281153

RESUMO

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.


Assuntos
Centrifugação/instrumentação , Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Animais , Centrifugação/métodos , Humanos , Microscopia/métodos , Manejo de Espécimes/métodos , Coloração e Rotulagem/métodos
5.
Molecules ; 26(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203841

RESUMO

Screening for systemic amyloidosis is typically carried out with abdominal fat aspirates with varying reported sensitivities. Fat aspirates are preferred for use in primary screening instead of organ biopsies as they are less invasive and thereby minimize the potential risk of complications. At Odense Amyloidosis Center, we performed a prospective study on whether the combined use of fat aspirate and tru-cut skin biopsy could increase the diagnostic sensitivity. Both fat aspirates and skin biopsies were screened with Congo Red staining, and positive biopsies were subsequently subtyped using immunoelectron microscopy and mass spectrometry. Seventy-six patients were included. In total, 24 patients had systemic amyloidosis (11 AL, 12 wtATTR, 1 AA), and 6 patients had localized amyloidosis. Combined fat aspirate and skin biopsy were Congo Red-positive in 15 patients (overall sensitivity (OS) 62.5%). Fat aspirates were positive in 14 patients (OS 58.3%), and the skin biopsy was positive in 5 patients (OS 20.8%). In only one patient did the skin biopsy add extra diagnostic information. The sensitivity differed between AL and ATTR amyloidosis-81.8% and 41.7%, respectively. Using skin biopsy as the only screening method is not recommended.


Assuntos
Proteínas Amiloidogênicas/análise , Amiloidose/diagnóstico , Amiloidose de Cadeia Leve de Imunoglobulina/diagnóstico , Tecido Adiposo/patologia , Adulto , Idoso , Amiloide/análise , Amiloidose/metabolismo , Biópsia/efeitos adversos , Feminino , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Estudos Prospectivos , Pele/patologia , Coloração e Rotulagem/métodos , Gordura Subcutânea/patologia
6.
Methods Mol Biol ; 2319: 61-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331243

RESUMO

The blood vascular system is a tree-like hierarchical branching structure and needs to function even before fully established. Abnormal formation of blood vessels results in embryonic lethality and also contributes to the pathogenesis of a number of human diseases, including cancer metastasis. To understand the molecular events associated with blood vessel formation, we established a fluorescence staining-based protocol on mouse embryonic skin. We harvested mouse embryonic skin and performed whole-mount staining. The reconstructed three-dimensional vascular structure provided detailed information on angiogenesis.


Assuntos
Células Endoteliais/citologia , Imuno-Histoquímica/métodos , Neovascularização Fisiológica , Pele/irrigação sanguínea , Pele/citologia , Coloração e Rotulagem/métodos , Animais , Células Endoteliais/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Pele/crescimento & desenvolvimento , Pele/metabolismo
7.
Methods Mol Biol ; 2319: 87-92, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331246

RESUMO

Blood vessel formation is a fine-regulated process and interfering with blood vessel formation causes embryonic lethality as well as associated with many diseases in the adult, including inflammatory, ischemic, and cancer metastatic diseases. Brain contains abundant blood vessels and has some unique physiological functions, such as blood-brain barrier. Due to the thickness and opaque characters of the tissues, it is a challenge to visualize the three-dimensional structures of the brain blood vessels in the mouse. Therefore, establishing a protocol to display the three-dimensional structures in the brain is required for exploring the regulatory molecular mechanisms in brain blood vessel formation. In this manuscript, we introduced a whole-mount and a vibratome thick section of mouse embryonic hindbrain to display the three-dimensional structures of brain vascular system.


Assuntos
Dissecação/métodos , Neovascularização Fisiológica , Rombencéfalo/irrigação sanguínea , Coloração e Rotulagem/métodos , Animais , Vasos Sanguíneos/diagnóstico por imagem , Vasos Sanguíneos/metabolismo , Dissecação/instrumentação , Células Endoteliais/metabolismo , Imunofluorescência , Camundongos , Microscopia Confocal , Rombencéfalo/crescimento & desenvolvimento , Rombencéfalo/metabolismo
8.
Methods Mol Biol ; 2319: 153-159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331253

RESUMO

Pathological alterations of lymphatic structure and function interfere with lymph transport, resulting in a wide range of clinical disorders that include edema, tissue inflammation, and metabolic syndromes. Mesentery contains abundant lymphatic vessels and plays an important role in transporting absorbed lipid from the intestine. In this manuscript, we describe a whole-mount staining method on isolated mouse mesentery with VEGFR3, Prox1, and Lyve1 antibodies to visualize the morphology of lymphatic vessels.


Assuntos
Linfangiogênese , Vasos Linfáticos/metabolismo , Mesentério/citologia , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Animais , Embrião de Mamíferos/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Mesentério/metabolismo , Camundongos , Proteínas Supressoras de Tumor/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo
9.
Molecules ; 26(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201280

RESUMO

Site-specific conjugation of proteins is currently required to produce homogenous derivatives for medicine applications. Proteins derivatized at specific positions of the polypeptide chain can actually show higher stability, superior pharmacokinetics, and activity in vivo, as compared with conjugates modified at heterogeneous sites. Moreover, they can be better characterized regarding the composition of the derivatization sites as well as the conformational and activity properties. To this aim, several site-specific derivatization approaches have been developed. Among these, enzymes are powerful tools that efficiently allow the generation of homogenous protein-drug conjugates under physiological conditions, thus preserving their native structure and activity. This review will summarize the progress made over the last decade on the use of enzymatic-based methodologies for the production of site-specific labeled immunoconjugates of interest for nuclear medicine. Enzymes used in this field, including microbial transglutaminase, sortase, galactosyltransferase, and lipoic acid ligase, will be overviewed and their recent applications in the radiopharmaceutical field will be described. Since nuclear medicine can benefit greatly from the production of homogenous derivatives, we hope that this review will aid the use of enzymes for the development of better radio-conjugates for diagnostic and therapeutic purposes.


Assuntos
Proteínas/química , Transglutaminases/química , Animais , Humanos , Imunoconjugados/química , Peptídeos/química , Coloração e Rotulagem/métodos
10.
Cell Physiol Biochem ; 55(S1): 171-184, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34156175

RESUMO

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.


Assuntos
Corantes/análise , Microglia/citologia , Pinocitose , Azul Tripano/análise , Animais , Morte Celular , Linhagem Celular , Sobrevivência Celular , Camundongos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos
11.
Nat Commun ; 12(1): 3648, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131146

RESUMO

Innovations in high-resolution optical imaging have allowed visualization of nanoscale biological structures and connections. However, super-resolution fluorescence techniques, including both optics-oriented and sample-expansion based, are limited in quantification and throughput especially in tissues from photobleaching or quenching of the fluorophores, and low-efficiency or non-uniform delivery of the probes. Here, we report a general sample-expansion vibrational imaging strategy, termed VISTA, for scalable label-free high-resolution interrogations of protein-rich biological structures with resolution down to 78 nm. VISTA achieves decent three-dimensional image quality through optimal retention of endogenous proteins, isotropic sample expansion, and deprivation of scattering lipids. Free from probe-labeling associated issues, VISTA offers unbiased and high-throughput tissue investigations. With correlative VISTA and immunofluorescence, we further validated the imaging specificity of VISTA and trained an image-segmentation model for label-free multi-component and volumetric prediction of nucleus, blood vessels, neuronal cells and dendrites in complex mouse brain tissues. VISTA could hence open new avenues for versatile biomedical studies.


Assuntos
Imageamento Tridimensional/métodos , Coloração e Rotulagem/métodos , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Feminino , Fluorescência , Imunofluorescência , Células HeLa , Humanos , Aprendizado de Máquina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Teóricos , Peixe-Zebra
12.
FEBS Lett ; 595(14): 1914-1919, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34080704

RESUMO

Biological structures with highly curved membranes, such as caveolae and transport vesicles, are essential for signal transduction and membrane trafficking. Although membrane proteins in these structures are subjected to physical stress due to the curvature of the lipid bilayers, the effect of this membrane curvature on protein structure and function remains unclear. In this study, we established an experimental procedure to evaluate membrane curvature-induced structural changes in the prototypical potassium channel KcsA. The effect of a large membrane curvature was estimated using fluorescently labeled KcsA by incorporating it into liposomes with a small diameter (< 30 nm). We found that a large membrane curvature significantly affects the activation gate conformation of the KcsA channel.


Assuntos
Proteínas de Bactérias/química , Lipossomos/química , Fosfatidilcolinas/química , Canais de Potássio/química , Potássio/química , Coloração e Rotulagem/métodos , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quimotripsina/química , Corantes Fluorescentes/química , Expressão Gênica , Transporte de Íons , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Rodaminas/química , Streptomyces coelicolor/genética
13.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070753

RESUMO

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.


Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Plantas/química , Genoma de Planta , Hibridização in Situ Fluorescente , Cebolas/genética , Coloração e Rotulagem/métodos , Cromossomos de Plantas/metabolismo , Sondas de DNA/síntese química , Sondas de DNA/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Ligação Genética , Marcadores Genéticos , Cebolas/metabolismo , Transcriptoma
14.
Methods Mol Biol ; 2325: 29-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34053048

RESUMO

During the last two decades, the immunology field has been focused on the study of conventional T-cells, leading to important advances in identification of specific subsets involved in promoting and suppressing immune response in patients with cancer, autoimmune disease or transplanted patients. In these recent years, the research on unconventional subset of CD4-CD8- double-negative T-cells is growing. DNTs are a unique subset of T-cells characterized by being CD4-CD8-CD3+ and express either ß or α T-cell receptors (TCR). In this chapter, we describe the methods used to phenotypically characterize and isolate these cells in order to study their functional profile.


Assuntos
Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Técnicas de Cultura de Células/métodos , Citometria de Fluxo/métodos , Linfócitos T Citotóxicos/metabolismo , Citocinas/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Fenótipo , Coloração e Rotulagem/métodos , Linfócitos T Citotóxicos/citologia
15.
Methods Mol Biol ; 2325: 107-124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34053054

RESUMO

Tissue microarray (TMA) is a smart technical innovation recently imposed in pathology research. This technology provides a high-throughput analysis of multiple tissues at the same time. The technique allows faster analysis and considerably reducing costs for the staining because many small representative tissue samples from hundreds of different cases are assembled on a single histologic slide. This versatile technique may improve conventional microscopic techniques to detect and characterize cytotoxic T lymphocytes (CTL). Immunohistochemistry (IHC) may be effectively employed in CTL characterization to identify the location and distribution of target antigens in tissues by staining with a specific antibody. The antibody may be conjugated to either a fluorescent or enzymatic label, and the location of the label seen through a microscope approximates the position of the target antigen.This article summarizes the technical aspects of tissue microarray construction and sectioning, advantages, application, and limitations associated with immunohistochemistry and immunofluorescence.


Assuntos
Imunofluorescência/métodos , Imuno-Histoquímica/métodos , Linfócitos T Citotóxicos/metabolismo , Análise Serial de Tecidos/métodos , Anticorpos , Antígenos , Corantes Fluorescentes , Humanos , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos
16.
Chem Soc Rev ; 50(10): 6240-6277, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34027939

RESUMO

Systematically dissecting the molecular basis of the cell surface as well as its related biological activities is considered as one of the most cutting-edge fields in fundamental sciences. The advent of various advanced cell imaging techniques allows us to gain a glimpse of how the cell surface is structured and coordinated with other cellular components to respond to intracellular signals and environmental stimuli. Nowadays, cell surface-related studies have entered a new era featured by a redirected aim of not just understanding but artificially manipulating/remodeling the cell surface properties. To meet this goal, biologists and chemists are intensely engaged in developing more maneuverable cell surface labeling strategies by exploiting the cell's intrinsic biosynthetic machinery or direct chemical/physical binding methods for imaging, sensing, and biomedical applications. In this review, we summarize the recent advances that focus on the visualization of various cell surface structures/dynamics and accurate monitoring of the microenvironment of the cell surface. Future challenges and opportunities in these fields are discussed, and the importance of cell surface-based studies is highlighted.


Assuntos
Microscopia de Fluorescência , Coloração e Rotulagem/métodos , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Humanos , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Engenharia Metabólica , Polissacarídeos/química , Polissacarídeos/genética , Polissacarídeos/metabolismo , Propriedades de Superfície
17.
Methods Mol Biol ; 2318: 241-254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019294

RESUMO

Cellular senescence plays a role in several physiological processes including aging, embryonic development, tissue remodeling, and wound healing and is considered one of the main barriers against tumor development. Studies of normal and tumor cells both in culture and in vivo suggest that MYC plays an important role in regulating senescence, thereby contributing to tumor development. We have previously described different common methods to measure senescence in cell cultures and in tissues. Unfortunately, there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers in order to assure the correct identification of senescent cells. Here we describe protocols for simultaneous detection of multiple senescence markers in situ, a quantitative fluorogenic method to measure senescence-associated ß-galactosidase activity (SA-ß-gal), and a new method to detect senescent cells based on the Sudan Black B (SBB) analogue GL13, which is applicable to formalin-fixed paraffin-embedded tissues. The application of these methods in various systems will hopefully shed further light on the role of MYC in regulation of senescence, and how that impacts normal physiological processes as well as diseases and in particular cancer development.


Assuntos
Senescência Celular/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Coloração e Rotulagem/métodos , Envelhecimento , Compostos Azo/química , Biomarcadores , Células Cultivadas , Senescência Celular/genética , DNA/genética , Corantes Fluorescentes/química , Genes myc/genética , Genes myc/fisiologia , Humanos , Naftalenos/química , Proteínas Proto-Oncogênicas c-myc/genética , beta-Galactosidase/análise , beta-Galactosidase/metabolismo
18.
Methods Mol Biol ; 2304: 37-64, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028710

RESUMO

Fluorescence microscopy is advantageous for investigating biological processes and mechanisms in living cells. One of the most important considerations when designing an experiment is the selection of an appropriate fluorescent probe. Equally important is deciding how the probe will be attached to the protein of interest. The advantages and disadvantages of different fluorescent probe types and their respective labeling methods are discussed to provide an overview on selecting appropriate fluorophores and labeling systems for fluorescence-based assays. Protocols are outlined when appropriate.


Assuntos
Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Animais , Células COS , Chlorocebus aethiops , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Nanopartículas , Imagem Individual de Molécula
19.
Nat Commun ; 12(1): 2650, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976192

RESUMO

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences.


Assuntos
Núcleo Celular/química , DNA/química , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Organelas/química , Animais , Arabidopsis/citologia , Benzimidazóis/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , DNA/genética , DNA/metabolismo , Fluorescência , Células HeLa , Humanos , Camundongos , Microscopia Confocal/métodos , Estrutura Molecular , Células NIH 3T3 , Organelas/metabolismo , Coloração e Rotulagem/métodos , Imagem com Lapso de Tempo/métodos
20.
Medicine (Baltimore) ; 100(20): e26004, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34011097

RESUMO

BACKGROUND: Early detection and diagnosis of high-grade cervical intraepithelial neoplasia grade 2 or higher (CIN2+) is critical for a good prognosis and appropriate treatment. The chief aim of our study was to evaluate the diagnostic performance of folate receptor-mediated staining solution detection (FRD) for CIN2+. METHODS: We conducted a systematic review and meta-analysis by searching the PubMed and EMBASE databases for studies published until May 2020, which assessed the diagnostic accuracy of FRD, human papilloma virus (HPV) testing, and ThinPrep cytology test (TCT) for the detection of CIN2+. Bivariate models were used to compare the diagnostic performance of FRD, HPV, and TCT. RESULTS: Six studies involving 2817 patients were included in this meta-analysis. The pooled specificity of FRD was higher than that of HPV and TCT for detecting CIN2+ (0.65, 0.12, and 0.39, respectively). The summary area under the receiver operating characteristic curve values using FRD, HPV, and TCT for detecting CIN2+ were 0.79, 0.95, and 0.77, respectively, indicating that FRD was superior to TCT. The diagnostic odds ratios of FRD, HPV, and TCT were 6 (95% CI: 5-7), 3 (95% CI: 2-5), and 3 (95% CI: 2-4), respectively, demonstrating that FRD had good diagnostic accuracy. CONCLUSION: FRD showed good diagnostic accuracy and higher specificity than HPV and TCT for detecting CIN2+. Based on our results, we propose that FRD could be a candidate for cervical screening, especially in underdeveloped countries.


Assuntos
Neoplasia Intraepitelial Cervical/diagnóstico , Detecção Precoce de Câncer/métodos , Infecções por Papillomavirus/diagnóstico , Coloração e Rotulagem/métodos , Neoplasias do Colo do Útero/diagnóstico , Alphapapillomavirus , Neoplasia Intraepitelial Cervical/patologia , Neoplasia Intraepitelial Cervical/virologia , Colo do Útero/patologia , Colo do Útero/virologia , Feminino , Receptor 1 de Folato/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Humanos , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal/métodos
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